The present invention relates to the control of gene expression. More particularly, this invention relates to the use of synthetic, modified oligonucleotides to down-regulate the expression of a gene in an animal.
The potential for the development of an antisense oligonucleotide therapeutic approach was first suggested in three articles published in 1977 and 1978. Paterson et al. (Proc. Natl. Acad. Sci. (USA) (1977) 74:4370-4374) discloses that cell-free translation of mRNA can be inhibited by the binding of an oligonucleotide complementary to the mRNA. Zamecnik et al. (Proc. Natl. Acad. Sci. (USA) (1978) 75:280-284 and 285-288) discloses that a 13 mer synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome inhibits RSV replication in infected chicken fibroblasts and inhibits RSV-mediated transformation of primary chick fibroblasts into malignant sarcoma cells.
These early indications that synthetic oligonucleotides can be used to inhibit virus propagation and neoplasia have been followed by the use of synthetic oligonucleotides to inhibit a wide variety of viruses, such as HIV (see, e.g., U.S. Pat. No. 4,806,463); influenza (see, e.g., Leiter et al. (1990) (Proc. Natl. Acad. Sci. (USA) 87:3430-3434); vesicular stomatitis virus (see, e.g., Agris et al. (1986) Biochem. 25:6268-6275); herpes simplex (see, e.g., Gao et al. (1990) Antimicrob. Agents Chem. 34:808-812); SV40 (see, e.g., Birg et al. (1990) (Nucleic Acids Res. 18:2901-2908); and human papilloma virus (see, e.g., Storey et al. (1991) (Nucleic Acids Res. 19:4109-4114). The use of synthetic oligonucleotides and their analogs as antiviral agents has recently been extensively reviewed by Agrawal (Trends in Biotech. (1992) 10:152-158).
In addition, synthetic oligonucleotides have been used to inhibit a variety of non-viral pathogens, as well as to selectively inhibit the expression of certain cellular genes. Thus, the utility of synthetic oligonucleotides as agents to inhibit virus propagation, propagation of non-viral, pathogens and selective expression of cellular genes has been well established.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucieotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007 discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Furdon et al. (Nucleic Acids Res. (1989) 17:9193-9204) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters in addition to either oligonucleotide phosphorothioate or methylphosphonate regions. Quartin et al. (Nucleic Acids Res. (1989) 17:7523-7562) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters and oligonucleotide methylphosphonates. Inoue et al. (FEBS Lett. (1987) 215:237-250) discloses chimeric oligonucleotides having regions of deoxyribonucleotides and 2′-O-methyl-ribonucleotides.
Many of these modified oligonucleotides have contributed to improving the potential efficacy of the antisense oligonucleotide therapeutic approach. However, certain deficiencies remain in the known oligonucleotides, and these deficiencies can limit the effectiveness of such oligonucleotides as therapeutic agents. For example, Wickstrom (J. Biochem. Biophys. Meth. (1986) 13:97-102) teaches that oligonucleotide phosphodiesters are susceptible to nuclease-mediated degradation, thereby limiting their bioavailability in vivo. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1990) 87:1401-1405) teaches that oligonucleotide phosphoramidates or methylphosphonates when hybridized to RNA do not activate RNase H, the activation of which can be important to the function of antisense oligonucleotides. Thus, a need for methods of controlling gene expression exists which uses oligonucleotides with improved therapeutic characteristics.
Several reports have been published on the development of phosphorothioate-linked oligonucleotides as potential anti-AIDS therapeutic agents. Although extensive studies on chemical and molecular mechanisms of oligonucleotides have demonstrated the potential value of this novel therapeutic strategy, little is known about the pharmacokinetics and metabolism of these compounds in vivo.
Several preliminary studies on this topic have been published. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1991) 88:7595-7599) describes the intravenous and intraperitoneal administration to mice of a 20 mer phosphorothioate linked-oligonucleotide. In this study, approximately 30% of the administered dose was excreted in the urine over the first 24 hours with accumulation preferentially in the liver and kidney. Plasma half-lives ranged from about 1 hour (t1/2α) and 40 hours (t1/2β), respectively. Similar results have been reported in subsequent studies (Iversen (1991) Anti-Cancer Drug Design 6:531-538; Iversen (1994) Antisense Res. Devel. 4:43-52; and Sands (1994) Mol. Pharm. 45:932-943). However, stability problems may exist when oligonucleotides are administered intravenously and intraperitoneally. More recently, Agrawal et al. reported that oligonucleotide hybrids containing 2′-O-methyl ribonucleotides at both the 3′- and 5′ ends and deoxyribonucleotide phosphorothioates in the interior portion were absorbed through the gastrointestinal (GI) tract of rats (Biochem. Pharm. (1995) 50:571-576).
Thus, there remains a need to develop more effective therapeutic methods of down-regulating the expression of genes which can be easily. manipulated to fit the animal and condition to be treated, and the gene to be targeted. Preferably, these methods should be simple, painless, and precise in effecting the target gene.